1. Field of the Invention
The present invention relates to a transformed microorganism producing an L-amino acid with a high yield using sucrose as a main carbon source, and a method for producing an L-amino acid using the same.
2. Background Art
Due to growing demand for bio-fuel production and crop failures caused by unusual climate, the price of starch sugar used as a fermentation feedstock has rapidly increased. Alternatively, the use of sucrose or molasses containing a high concentration of sucrose, cheaper than starch sugar, as a carbon source in industrial fermentation, is advantageous to ensure the cost competitiveness. Approximately 50% of the E. coli isolated from nature is able to metabolize sucrose, but E. coli K12 strain, B strain and C strain usually used in industrial fermentation, have no ability to assimilate sucrose (Mol. Microbiol. (1998) 2:1-8, Can. J. Microbiol. (1999) 45:418-422). Therefore, among the most important challenges in the fermentation industry is the investigation of genes involved in sucrose assimilation, the establishment of enhanced sucrose assimilation-related genes by improvement, and the application of the genes to the sucrose non-assimilative, industrial E. coli strains for the production of desired metabolites.
To impart sucrose-assimilating ability to industrial E. coli strains, methods of introducing a sucrose assimilation gene or gene cluster derived from microorganisms having a sucrose-assimilating ability have been generally used. For example, a method of imparting sucrose-assimilating ability to E. coli K12 by transformation with the scr regulon present in the species Salmonella belonging to the family Enterobacteriaceae (J. Bacteriol. (1982) 151:68-76, Mol. Microbiol. (1998) 2:1-8, J. Bacteriol. (1991) 173:7464-7470, U.S. Pat. No. 7,179,623), Klebsiella pneumoniae (J. Gen. Microbiol. (1988) 134:1635-1644), Erwinia amylovora (J. Bacteriol. (2000) 182:5351-5358) has been well known in the art. Introduction of the csc regulon derived from non-K12 E. coli having the sucrose-assimilating ability or pathogenic E. coli (Appl. Environ. Microbiol. (1992) 58:2081-2088, U.S. Pat. No. 6,960,455), introduction of sucrose assimilation gene that is present in conjugative plasmid scr53 isolated from E. coli AB1281 (U.S. Pat. No. 4,806,480), and introduction of scr regulon and sac operon derived from Gram-positive microorganisms, Streptococcus mutans (J. Bacteriol. (1989) 171:263-271) and Bacillus subtilis (J. Bacteriol. (1989) 171:1519-1523) are also known.
Conventionally, L-amino acid has been industrially produced by fermentation methods using microorganism strains isolated from nature or artificial mutants of said bacterial strains, which have been modified in such a way that the L-amino acid production yield is enhanced. Many techniques to enhance L-amino acid production yields have been reported. For example, techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid. Meanwhile, amino acid production yields of L-amino acid-producing strains can be improved by enhancing L-amino acid excretion activity. For example, bacteria belonging to the genus corynebacterium in which expression of an L-lysine secretion gene is increased have been used. In addition, expression of genes coding for the efflux proteins which act to enhance secretion of L-cysteine, L-cystine, N-acetylserine, or thiazolidine derivatives is regulated to increase L-amino acid production activity.
The sucrose utilization system is largely divided into the Scr-PTS system and the Scr-non PTS system. Most microorganisms capable of utilizing sucrose have the Scr-PTS (phosphoenolpyruvate dependent sucrose phosphotransferase) system. The Scr-PTS system allows efficient uptake of a low level of sucrose, but the sucrose uptake process requires PEP (phosphoenolpyruvate) consumption to reduce the intracellular PEP pool. The Scr-non PTS system is a system using proton symport-type sucrose permease, exemplified by the well known csc gene clusters containing cscB coding for sucrose permease. csc regulon consists of cscB (sucrose permease or proton symport-type sucrose permease), cscK (fructokinase), cscA (sucrose hydrolase), and cscR (sucrose transcriptional regulator), and is negatively controlled by two operons, cscKB and cscR (Jahreis K et al., J. Bacteriol. (2002) 184:5307-5316).
PEP is a key metabolite in the central metabolic pathway, and functions as a phosphate donor of the sugar PTS system. PEP is also involved in ATP synthesis catalyzed by pyruvate kinase, and functions as a direct precursor of several amino acids or oxaloacetate (OAA) (Metab. Eng. (2002) 4:124-137, Microb. Cell Fact. (2005) 4:14). In particular, OAA is used as a carbon skeleton of amino acids such as threonine, isoleucine, methionine, lysine, asparagine and aspartic acid (U.S. Pat. No. 6,960,455). PEP is known to be mostly consumed via the sugar PTS system. Up to 50% of the total PEP is consumed via the glucose PTS system in a minimal medium containing glucose as a carbon source (Microb. Cell Fact. (2005) 4:14). Therefore, if using a sugar non-PTS system instead of sugar PTS system, intracellular PEP pool can be increased, and the increased PEP can be used for biosynthesis of fermentation products, thereby improving productivity and yield.
In sucrose utilization, the Scr-non PTS system, which requires no PEP consumption upon sucrose uptake, is also more preferred than Scr-PTS system. Practically, Ajinomoto Co. has introduced methods of making threonine, isoleucine and tryptophan using E. coli transformed with EC3132-derived cscBKA (U.S. Pat. No. 6,960,455). DuPont has also produced tyrosine using E. coli K12 transformed with E. coli ATCC13281-derived cscBKAR and sucrose (Appl. Microbiol. Biotechol. (2007) 74:1031-1040).
Meanwhile, mannokinase (Mak) has a kinase activity that converts hexose, including mannose and fructose, to 6-phospho-ester by ATP consumption. In particular, a gene (mak or yajF) encoding the wild-type Mak (Mak-o) of enteric bacteria is known as a cryptic gene, and its activity is greatly increased by sequence mutation in the promoter-35 region of mak (Mak+) (Kornberg H L, J. Mol. Microbiol. Biotechnol. (2001) 3:355-359; Miller B G & Raines R T, Biochemistry (2005) 44:10776-10783). Mak is also able to phosphorylate other substrates such as glucose, sorbose, and glucosamine, in addition to mannose and fructose. However, there have been no reports that Mak affects sucrose metabolism.